| Background and ObjectiveThe most frequent causes of male infertility are low sperm concentration(oligospermia) and reduced sperm motility(asthenospermia). These two semen disorders also frequently occur in combination, a condition known as oligoasthenozoospermia. While the effects of such conditions on the process of fertilization are well known(i.e. reduced likelihood of reaching the egg in the oviduct), the causative mechanisms remain largely unknown.Extensive research efforts to elucidate the pathogeneses of oligospermia and asthenospermia have suggested roles for chromosome abnormalities, perturbed gene regulation, environmental factors, infection- and immune-related factors, and endocrine dysfunction. Molecular studies have also demonstrated that the mitochondrial pathway of apoptosis plays a key role in male infertility. To date, however, the majority of studies of apoptosis factors have assessed sperm in the testicular tissue; studies of ejaculated spermatozoa, especially those from male patients diagnosed with oligospermia and/or asthenospermia, are rare.An important component of mitochondria-dependent apoptosis is the crosstalk between oxygen free radicals and various apoptosis-inducing factors. Intriguingly, a substantial proportion(up to 40%) of men diagnosed with semen disorders show high levels of ROS. Many common environmental factors are known to stimulate ROS production, and are introduced by routine medical care(i.e. synthetic hormones and antibiotics), the workplace environment(plasticizers and ionizing radiation), and lifestyle practices(tobacco smoking and alcohol consumption). Furthermore, various studies of oligospermia and asthenospermia cases have demonstrated a close association between the conditions and these environmental factors. The pathogenic potential of stimulated ROS has also been shown in some disease conditions(e.g. traumatic brain injury and myocardial ischemia) and involved in triggering of the mitochondrial apoptotic pathway.The current study was designed to investigate the potential role of oxygen free radicals and the mitochondrial-dependent apoptosis pathway as a pathogenic mechanism of oligospermia, asthenospermia, and oligoasthenozoospermia. The malondialdehyde content(MDA), the activities of total superoxide dismutase(T-SOD) and glutathione peroxidase(GSH-Px) were determined for the study of oxygen free radicals. The m RNA and protein expression levels of apoptosis-related genes(Bcl-2, Bax, Cytochrome C and Caspase-3) were measured for mitochondrial signaling investigation.Then, through respective incubation of glutathione and exogenous Cyt C in each group of semen, and by the TBA method to measure changes in MDA, and the Real-time PCR method to observe pathway related indicators, we analyzed the two drugs’ targets in this pathway to oligospermia and asthenospermia.Finally, after constructed vectors by chemical synthesis of Bax-si RNA were transfected into mouse testieular cell line(TM4 cells), we analyzed the expression varieties of Bax and the other genes of mitochondrial pathway.Methods and methodsPart 1:(1)The content of MDA and the activity of T-SOD and GSH-Px were determined by enzymatic assays. The mitochondrial membrane potential(MMP) was determined by flow cytometry detection of accumulated membrane-permeable JC-1 fluorescent dye. It was the computer-assisted semen analysis(CASA) system that determined sperm motility parameters: sperm motility, curve speed(VCL), linear velocity(VSL), average path velocity(VAP), and amplitude of lateral head displacement(ALH). The m RNA expression levels of apoptosis-related genes(Bcl-2, Bax, Cyt C, and Caspase-3) were measured by Real-time PCR.(2)The content of MDA and the activity of T-SOD and GSH-Px were determined by enzymatic assays.The protein expression levels of apoptosis-related genes(Bcl-2, Bax, Cyt C, and Caspase-3) were measured by Immunocytochemistry and Western blotting.Part 2:Collecting normal semen by the packet, adding hypoxanthine, xanthine oxidase system to construct semen oxidative damage model, adding different concentrations of GSH simultaneously, after 2 h and 12 h incubation, the content of MDA and activity of T-SOD and GSH-Px were determined by enzymatic assays; the sperm motility parameters were determined by the CASA system, including sperm motility, VCL, VSL, VAP, and ALH; the m RNA expression levels of apoptosis-related genes(Bcl-2, Bax, Cyt C, and Caspase-3) were measured by Real-time PCR.Part 3:Normal semen and asthenospermia semen were collected by the packet, added by different concentrations of exogenous Cyt C. After 2 h and 12 h incubation, the content of MDA and activity of T-SOD and GSH-Px were determined by enzymatic assays. The CASA system determined sperm motility parameters: sperm motility, VCL, VSL, VAP, and ALH. The m RNA expression levels of apoptosis-related genes(Caspase-3) were measured by Real-time PCR.Part 4:Four different si RNA sequences were designed according to the mouse Bax m RNA target sequence, Bax-si RNA1, Bax-si RNA2, Bax-si RNA3 and Bax-si RNA4, which were transfected into competent cells by construction of a vector. They were taken as blank control group(without transfection), negative control group, Bax-si RNA(167) group, Bax-si RNA(283) group, Bax-si RNA(408) group and Bax-si RNA(514) group, respectively. The expression of Bax m RNA were detected using Real-time PCR technology. After comparison and analysis of the different expression of Bax, the effective sequence of si RNA was identified, including Bax-si RNA(167) and Bax-si RNA(283). Then, the two effective sequence were transfected into TM4 cells; consequently, Bax, Bcl-2, Cyt C and Caspase-3 m RNA were detected with Real-time PCR at various time points 48 h and 72 h after transfection to observe the interfering effects of si RNA.ResultsPart 1:1. All groups with semen disorders had significantly low T-SOD and GSH-Px activities(all P<0.05 vs. normal sperm), and the extent of reduction showed a disorder-related trend(asthenospermia < oligospermia ≈ oligoasthenozoospermia). In contrast, the patients with semen disorders had significantly higher MDA content(all P<0.05 vs. normal sperm), and the extent of increase showed a disorder-related trend(asthenospermia > oligospermia ≈ oligoasthenozoospermia).2. The sperm from patients with semen disorders also showed significantly lower MMP(as evidenced by lower mean ratios of JC-1+ sperm per group).3. All groups with semen disorders had significantly higher m RNA and protein expression levels of Bax, Cyt C and Caspase-3 but significantly lower levels of Bcl-2(all P<0.05 vs. normal sperm). Only the extent of increases in Cyt C and Caspase-3 showed a disorder-related trend(oligospermia > asthenospermia).4. Compared with normal control group, in addition to VCL, VSL and VAP in oligozoospermia group, the motion parameters of other groups were all significantly decreased(P<0.05).Part 2:1. Compared with group A(the control group), MDA content increased in group B(plus oxidase reaction system), group C(plus oxidase reaction system, a low concentration of GSH) and group D(plus oxidase reaction system, a high concentration of GSH)(P<0.01, P<0.05, P<0.05, respectively). Compared with group B, MDA levels were lower in group C and group D(all P<0.05).2. Compared with group A, T-SOD and GSH-Px activity decreased in group B, and group C. Compared with group B, T-SOD and GSH-Px activity increased in group C, group D(P<0.05, P<0.01, respectively).3. Compared with group A, sperm motion parameters were reduced in group B, and group C(P<0.01). Compared with group B, sperm motion parameters were increased in group C, and group D(P<0.05).4. Compared with group A, sperm Bcl-2 expression was reduced but expression of Bax, Cyt C and Caspase-3 was increased in group B, and group C. Compared with group B, sperm relative expression of Bax, Cyt C and Caspase-3 in group D was lower, significantly(P <0.05).Part 3:1. Compared with group A1(the control group of normal semen), MDA contents were decreased in group B1(plus a low concentration of exogenous Cyt C), group C1(plus intermediate concentration of exogenous Cyt C), and group D1(plus high concentration of endogenous Cyt C). Compared with group A2(the control group of asthenospermia semen), MDA contents were decreased in group B2(plus a low concentration of exogenous Cyt C), group C2(plus Intermediate concentration of exogenous Cyt C), and group D2(plus high concentration of endogenous Cyt C). MDA levels were significantly lower in group C2 and group D2 than in group B2(P<0.05).2. Compared with that in group A1, T-SOD and GSH-Px activity was significantly increased in group D1(P<0.05), and in comparision with that in group A2, T-SOD and GSH-Px activity was increased significantly in group C2 and group D2(P<0.05, P<0.01, respectively).3. Compared with those in group A1, sperm motion parameters showed a gradual increasing trend in group B1, group C1 and group D1, without a significant difference(P>0.05). Compared with group A2, sperm motion parameters showed a gradual increasing trend in group B2, group C2 and group D2. Sperm vigor and rapid former sport increased significantly in group C2 and group D2, respectively(P<0.05).4. Compared with group A1, the expression levels of sperm Caspase-3 were significantly increased in group C1 and group D1,(P<0.01, P<0.05, respectively), and when compared with those in group A2, the expression levels of sperm Caspase-3 were significantly increased in group D2(P<0.05).Part 4:Two kinds of Bax-si RNA transfected into TM4 cells for 48 h and 72 h, the expressions of Bax, Cyt C and Caspase-3 gene m RNA were decreased, while those of Bcl-2 gene m RNA was increased, compared with those of the blank control group(P<0.05). However, the expression level did not change much in the negative control group. The interference effect of Bax-si RNA2 sequence was better than that of Bax-si RNA1 sequence. The interference effect of the time point(48 h) was better than that of the time point(72 h).Conclusion1. Oxygen free radicals could play a role in reducing sperm concentration and motility, possibly through effects on the mitochondrial apoptosis signaling pathway. Perturbed mitochondrial release of Cyt C might be the distinguishing molecular feature between oligospermia and asthenospermia.2. ROS and GSH were responsible for sperm’s damage and protection, respectively, in particular, reflecting the changes in motion parameters. ROS was able to effect on semen parameters related to oxygen free radicals- mitochondrial signaling pathway. GSH affected ROS directly, and showed certain dose-effect relationship.3. Exogenous CytC can perform an antioxidant reaction in seminal plasma of normal semen and asthenospermia, play a role in improving sperm motility in asthenospermia, play a part in increasing the expression of Caspase-3 in normal semen using a high concentration of Exogenous Cyt C, while in the case of asthenospermia the role is significantly low. Therefore, Exogenous Cyt C could ameliorate the asthenospermia.4. The effective sequence of si RNA(Bax-si RNA1 and Bax-si RNA2) targeting Bax gene was screened from four candidates. When transfected into TM4 cells, they could interfere the expression of Bax, Bcl-2, Cyt C and Caspase-3. Bax-si RNA2 can exert a better jamming effect after transfection of 48 h. |
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